One type of process utilized for the detection of hybridized nucleic acids involves polymerase chain reaction (PCR). The PCR process is well known in the art (U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159). In PCR, nucleic acid primers that are complementary to opposite strands of a nucleic acid amplification target sequence are permitted to anneal to the denatured sample. Next, DNA polymerase (typically heat stable) extends the DNA duplex from the hybridized primer. The process is then repeated to amplify the nucleic acid target. If the nucleic acid primers do not hybridize to the sample, then there is no corresponding amplified PCR product. In this case, the PCR primer acts as a hybridization probe.
In a PCR method, the amplified nucleic acid product may be detected in a number of ways, e.g., by incorporation of a labeled nucleotide into the amplified strand by using labeled primers. Primers used in PCR include, but are not limited to radioactive substances, fluorescent dyes, digoxygenin, horseradish peroxidase, alkaline phosphatase, acridinium esters, biotin and jack bean urease. PCR products made with unlabeled primers may be detected in other ways, such as electrophoretic gel separation followed by dye-based visualization.
PCR-based methods are of limited use for the detection of nucleic acids of unknown sequence. The human genome is composed of about 3×109 nucleotides; thus, it is difficult to isolate and analyze a specific human gene. Yet, PCR can amplify a target sequence with high speed, specificity and sensitivity by using a set of primers including primers complementary to both ends of the target sequence (Saiki et al. Science 239: 487, 1988).
PCR may be widely used in analyzing a disease-associated gene. Specifically, gene amplification by PCR may be useful for analyzing genetic variations of a disease-associated gene in the medical field. A specific disease-associated gene may be amplified using PCR, and analyzed by using a sequencing, hybridization or single strand conformational polymorphism. In analyzing genetic variations of a gene a single PCR may be enough to amplify the entire gene if the size of a target gene is small. However, if the size of a target gene is large, e.g., 1 kb or more, a single PCR may have difficulty in amplifying the entire gene. Thus, PCR may be conducted several times on several portions of the entire gene to amplify the entire gene of a large target gene. In analyzing a genetic variation of a disease-associated gene, a multiple PCR is more frequently used than a single PCR since most disease-associated genes may be 1.5 kb or larger in size.
A multiple PCR process requires a large amount of a sample, for example, a patient's DNA or blood. A multiple PCR also costs more and requires more effort and time. Thus, multiplex PCR assays have been developed to solve the above problems. A multiplex PCR simultaneously amplifies a plurality of target sequences of a gene in one reaction. Therefore, a plurality of target sequences are amplified by a single PCR using a primer pool for amplifying each target sequence.
Multiplex PCR assays are well known in the art. For example, U.S. Pat. No. 5,582,989 discloses the simultaneous detection of multiple known DNA sequence deletions. The technique disclosed therein uses a first set of probes to hybridize to the targets. Those probes are extended if the targets are present. The extension products are amplified using PCR.
A set of primers for a multiplex PCR may be able to specifically bind to a target sequence and should not interfere with each other in order to amplify the target sequence by a sufficient amount. A multiplex PCR using such a set of primers may be able to save time, effort and cost for amplifying a target sequence in comparison with a single PCR. In analyzing a genetic variation of a gene by using a DNA chip, a multiplex PCR may be useful in amplifying more than one kind of DNA sample in a reaction. Such a DNA chip may be useful in analyzing genetic variations in a gene.
It is known that a genetic variation of a human glucokinase gene, including a point mutation, causes maturity-onset diabetes in the young (MODY 2) (Matschinsky & Magnuson, in ‘Molecular Pathogenesis of MODYs’, Karger, 1998; U.S. Pat. No. 5,541,060; WO9321343). MODY 2, a kind of MODY disease (MODY 1, 2, 3, 4 and 5) accounts for about 10–30% of all type II diabetes mellitus cases. Thus, in analyzing a genetic variation of a human glucokinase gene, it is possible to anticipate a person's propensity to a diabetes mellitus. Therefore, in order to rapidly analyze a human glucokinase gene using, for example, a DNA chip, a set of primers for amplifying a human glucokinase gene by a multiplex PCR needs to be developed.